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Original Studies |
Anatomisches Institut, Technische Universität München (S.F., S.B., A.M.), D-80802 Munich; Abteilung für Allgemeine Physiologie, Universität Ulm (K.J.F.), D-89069 Ulm; and I. Frauenklinik der Ludwig-Maximilians-Universität München (U.B., C.B.), D-80337 Munich, Germany
Address all correspondence and requests for reprints to: Artur Mayerhofer, M.D., Anatomisches Institut, Technische Universität, Biedersteiner Strasse 29, D-80802 Munich, Germany. E-mail: mayerhofer{at}lrz.tu-muenchen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We speculated that the receptor subtype mediating the effects should be of the M1, M3, or M5 (since then described) type, which are all linked to phosphoinositol mobilization and to increases in intracellular free Ca2+ concentrations (3, 4). However, the precise nature of the receptor is not known. A functional GC muscarinic receptor was identified by Morely et al. (5) using Ca2+ measurements in another species, chicken, but likewise, the precise nature of the receptor type has not been identified. Muscarinic receptors linked to oxytocin and progesterone production have been described in luteinized bovine GC (6), but we (human GC) and Morley et al. (chicken GC) (5) did not find evidence for involvement of this receptor in GC steroid production, because carbachol did not affect basal or hCG-stimulated progesterone or estradiol production (Mayerhofer, A., et al., unpublished). Thus, until the present time neither the muscarinic receptor subtype in human GC nor its possible function(s) was clear.
The natural ligand of muscarinic receptors is ACh. A potential physiological source of this neurotransmitter is the cholinergic nerve fibers reported to innervate the ovary (7, 8). Cholinergic fibers in the ovary have been reported at the ultrastructural level to contain small electron-translucent, clear vesicles, the presumed storage organelles for ACh. However, few of these fibers are found in the ovary, especially compared to the fibers with electron-dense granules representing the catecholaminergic and peptidergic innervation (7, 8, 9, 10, 11). Peptidergic and catecholaminergic innervation was furthermore demonstrated using antibodies to neuropeptides and catecholamine-synthesizing enzymes (8). In contrast, to date mainly histochemical methods visualizing ACh esterase, the enzyme involved in the breakdown of ACh, were used to determine ACh-containing nerve fibers in the ovary. It is doubtful whether this marker is indeed a good indicator for the presence of ACh (8). Thus, although cholinergic nerve fibers are a possible source of ACh, which subsequently may act on GC, the relatively low number of cholinergic fibers reported to be present in the ovary may not favor the idea of a major supply from this source. Whether ACh could be derived from another intragonadal source is not known. The expression of the synthesizing enzyme choline-acetyltransferase (ChAT) in the testis (12) and placenta (13, 14, 15), however, nourished our speculation about a possible local ACh source within the ovary.
In the present study we have therefore attempted to clarify the precise nature of the muscarinic receptor of human GC and its possible function. We have also addressed the issue of a possible intraovarian production of ACh by investigating whether the gene for ChAT, the enzyme responsible for ACh synthesis, and the gene for the vesicular ACh transporter (VAChT), which is responsible for uptake of ACh in vesicles, are expressed by human GC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Follicular fluid containing granulosa cells was derived from in vitro fertilization patients. Isolation and culture of the cells were performed as described (1, 16, 17, 18, 19, 20). The experimental procedure and use of cells were approved by the local ethics committee (1, 20). All culture dishes were coated with laminin, as previously described (20). Cultures were kept in an incubator at a humidified atmosphere with 5% CO2 at 37 C. After 24 h media were replaced, and nonadherent cells were removed by gentle washing. At that time stimulation experiments were started and lasted for the next 24 h. The following substances were added: carbachol (Carb; 0.1 µmol/L, a stable acetyl+choline derivate; Sigma Chemical Co., Deisenhofen, Germany), hCG (10 IU/µL; Sigma Chemical Co.), pirenzepine dihydrochloride (Pir; 1–10 µmol/L; a selective M1 muscarinic antagonist; Biotrend, Köln, Germany), or oxotremorine M (Oxo; 1 µmol/L; a muscarinic acetylcholine agonist; Biotrend). Concentrations were chosen to match the ones described previously (21). Cells were harvested for ribonucleic acid (RNA) extraction (for subsequent RT-PCR), protein analysis in Western blots, immunocytochemistry, or proliferation assays as described below.
Calcium measurements in single cells
Fura-2 and fura-2/AM (pentaacetoxymethyl ester) were obtained from Calbiochem (La Jolla, CA), and dimethylsulfoxide (DMSO) was purchased from Fluka (Neu Ulm, Germany). As reported (1), cells were loaded with fura-2/AM in culture medium without serum (15–30 min at 37 C). Fura-2/AM was added from a stock to give a final concentration of 1.5 µmol/L and 0.1% dimethylsulfoxide. The cells were rinsed with solution A (140 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 1 mmol/L CaCl2, 0.5 mmol/L ascorbic acid, 11 mmol/L glucose, and 15 mmol/L PIPES, pH 7.2) and used for Ca2+ measurements within 1–4 h after dye loading. Fluorescence measurements were performed with the Zeiss Microscope Photometer System (FFP, Oberkochen, Germany), as described previously (1, 16, 17, 18, 19). Ratios of the recordings at 340–380 nm were determined (1, 16, 17, 18, 19). All test substances were dissolved in solution A. Drugs were applied with the L/M-SPS-8 superfusion system (List, Darmstadt, Germany), which allows repetitive and highly reproducible drug applications to a single cell or a small group of cells in a repetitive and highly reproducible manner. To restrict the drug action to a small volume within the petri dish, we established a combination of two bath perfusion systems. A global bath perfusion with the inflow set to 1.5 mL/min and an outflow that removed excess fluid. With the local perfusion system, a continuous fluid stream was generated into which the drugs were released. The entry (tip of a multibarreled drug application pipette) was positioned at a distance of about 50–100 µm upstream and the outflow at about 300 µm downstream to the measuring field. The local outlet setting of 1 mL/min almost guaranteed a complete removal of the local inflow. The selection between the various supply vessels was controlled by magnetic valves. Pressure onto the supply vessels was adjusted using the MPCU-3 system (Lorenz, Lindau, Germany). The concentrations reported in the following are undiluted concentration in the syringe (stock) before adding them to the cells.
RT-PCR cloning and Western blotting
In pilot RT-PCR experiments we did not find evidence for M3 receptor expression by human GC. Therefore, we focused in the present study on the M1 receptor. Total RNA from human GC was prepared using a commercial RNA extraction kit (RNEasy, Qiagen, Hilden, Germany). We used 100–300 ng total human GC RNA for RT using a 18-mer polydeoxythymidine primer and Moloney’s murine leukemia virus reverse transcriptase (Stratagene, Heidelberg, Germany) (22). Primers used for PCR were: 18-mer, M1 receptor 5'-sense primer, 5'-AGC TCC CCA AAT ACA GTC-3', complementary to nucleotides (nt) 1061–1078 of human M1 receptor messenger RNA (mRNA) sequence (GenBank accession no. X15263 and X13530) (23); and 3'-antisense primer, 5'-TTG CAG AGT GCG TAG CAG-3', complementary to nt 1348–1365; and VAChT 5'-sense primer, 5'-ACG TGG ATG AAG CAT ACG-3' complementary to nt 2022–2039 of human VAChT (GenBank accession no. U10554) (24); and 3'-antisense primer, 5'-CTG AGA CAT GGC GCA CGT-3' complementary to nt 2311–2328. The PCR reactions were carried out in a PTC-200 Peltier Thermal Cycler (Biozym, Hessisch Oldendorf, Germany) using Taq polymerase (Promega Corp., Heidelberg, Germany) as previously described (22, 25). PCR amplification consisted of 35 cycles of denaturing (94 C, 1 min), annealing (55 C, 1 min), and extension (72 C, 1 min). The PCR reaction products were separated on 2% agarose gels and visualized with ethidium bromide. To identify the M1 receptor and VAChT complementary DNAs (cDNAs), they were subcloned into the pGEMT vector (Promega Corp.). cDNA clones containing the insert were sequenced using a fluorescence-based dideoxy sequencing reaction (Prism Ready Reaction Dye Terminator Cycle Sequencing Kit) and Ampli-Taq DNA polymerase. Automated sequence analysis was performed on an ABI model 377 DNA sequencer (Perkin Elmer, Überlingen, Germany) (20, 26).
Western blotting was performed as previously described in detail (20, 27). Cells were harvested in a buffer containing 150 mmol/L NaCl, 10 mmol/L PIPES, and 1 mmol/L ethylenediamine tetraacetate, pH 7.2, and frozen at -20 C. Samples were thawed, homogenized in 62.5 m mol/L Tris-HCl buffer (pH 6.8) containing 10% sucrose and 2% SDS, sonicated, and heated in the presence of ß-mercaptoethanol (95 C for 5 min). Samples (15 µg protein/lane; bicinchoninic acid method, Pierce Chemical Co., Oud Beijerland, The Netherlands) were separated electrophoretically on 12% or 10% SDS-polyacrylamide gels. Proteins were blotted onto nitrocellulose membranes and probed with antisera/antibodies directed against VAChT (goat antivesicular acetyl-choline transporter antiserum generated against the C-terminal synthetic peptide sequence corresponding to amino acids 511–530 from the cloned rat VAChT; 1:1000; Sigma Chemical Co.), anti-ChAT (monoclonal mouse antihuman choline-acetyltransferase antibody; 1:500 to 1:1000; Boehringer Mannheim, Mannheim, Germany),
Proliferation studies
To quantitate cell proliferation a commercial assay for colorimetric determining the number of viable cells (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega Corp.) was used. Assays were performed by adding 20 µL reagent directly to culture wells and were incubated for 1 h, then absorbance was recorded at 490 nm in an enzyme-linked immunosorbent assay plate reader (Dynex, Denkendorf, Germany) using a reference wavelength of 630 nm to reduce background. The quantity of formazan product built by bioreduction is directly proportional to the number of living cells in culture.
Statistics
Student’s t test or ANOVA was used to evaluate the data.
Immunocytochemistry
The cellular distribution of M1 receptor, VAChT, and ChAT proteins in cultured human GC was determined by immunocytochemistry, using commercially available polyclonal antisera or monoclonal antibodies, two of which were also used for Western blotting (see above). Cultured cells (in Lab-Tek plates, Nunc, Copenhagen, Denmark) were fixed with Zamboni (2% paraformaldehyde and 15% picrinic acid, pH 7.3) or 4% paraformaldehyde in 0.05 mol/L phosphate-buffered saline, pH 7.4. Immunocytochemical procedures for detection of VAChT and ChAT using the avidin-biotin complex method were performed as previously described (28). Incubation with the antiserum/antibody was carried out at 4 C overnight in a humidified chamber. Biotinylated secondary antisera, goat antirabbit IgG, goat antimouse, and donkey antigoat, 1:500 diluted in phosphate-buffered saline with 1% BSA (Camon, Wiesbaden, Germany) and a commercial ABC kit (Vectastain, Camon) were subsequently used. The immunoreaction was visualized with 0.01% H2O2 and 0.05% diaminobenzidene solution (in 0.05 mol/L Tris-HCl, pH 7.6). Immunoreactions for M1 receptor detection were performed using an indirect fluorescence method, as previously reported (26), and a fluorescein isothiocyanate-labeled antirabbit IgG secondary antiserum (affinity purified with minimal cross-reactivity to human serum, Jackson ImmunoResearch Laboratories, Inc., Dianova, Hamburg, Germany). For control purposes the first antiserum was omitted, or incubation with nonimmune normal rabbit, mouse, or goat serum (1:5000 to 1:3000) was instead carried out. Sections were examined with a Zeiss Axiovert microscope, which was also equipped with a fluorescein isothiocyanate filter set. A Leica Corp. TCS NT confocal laser scanning microscope (Heidelberg, Germany) was used to examine cultured human GC cells immunostained with an M1 receptor antiserum (rabbit antihuman M1 receptor antiserum; 1:800; Biotrend) as well as control cells (rabbit nonimmune antiserum; 1:500). Some of the cells were counterstained with propidium iodide (Sigma Chemical Co.; 2 µg/mL in water for 2 min) to localize the nucleus as previously described (29, 30). Up to eight optical sections were collected and projected. Digital images were labeled using Photoshop (Adobe Systems, Mountain View, CA) and Freehand 5.0 (Macromedia, Inc., San Francisco, CA). Color prints were produced using an Epson Stylus Photoex (Düsseldorf, Germany) printer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using RT-PCR and oligonucleotide primers corresponding to the M1
receptor sequence, we obtained from reverse transcribed human GC mRNA
cDNAs of 304 bp (Fig. 1A
), which were
subcloned and sequenced. Analysis of the sequence data (two clones from
human GC) showed that they were identical to the human M1 receptor
sequence.
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). Controls with rabbit normal serum or controls with omission of the
M1 receptor antiserum were negative (Fig. 1C and not shown). The human GC M1 receptor is functionally linked to intracellular Ca2+ and to proliferation of human GC
Our previous single cell measurements (1) had shown that Carb in
the range from 10 nmol/L to 1 mmol/L induced a transient increase in
intracellular free Ca2+ in human GC. We therefore tested in
the present study whether the actions of the muscarinic agent Oxo at
concentrations that produce a reliable, substantial Ca2+
signal namely at 100 µmol/L, can be antagonized by the M1 receptor
antagonist Pir (10 and 100 µmol/L; Fig. 2A). The Ca2+ signal was
either completely abolished or substantially reduced (n = 7; Fig. 2B) when Pir at 10 µmol/L was used. The degree to which Pir was
affected appeared to depend on the maximal Ca2+ increase;
thus, if large Ca2+ transients were seen, Pir was not able
to completely antagonize the Oxo effect. However, Ca2+
signals induced by the agonist were always completely blocked using
pretreatment of equimolar concentrations of Pir, thus indicating that
the observed Ca2+ signal is a consequence of M1 receptor
activation.
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). Proliferation was significantly
increased after 24 h of treatment with 0.1 µmol/L Carb
(148.8 ± 6.7% over the control value). Pir (1–10 mmol/L) alone
slightly, but not significantly, reduced the cell proliferation rate.
However, Pir at 1 and 10 µmol/L completely prevented Carb-induced
cell proliferation.
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Both immunocytochemical staining of fixed human GC and Western
blotting using a monoclonal antibody against ChAT (Fig. 4A) indicated that the ACh-synthesizing
enzyme is present in human GC. Thus, the majority of the cells showed
cytoplasmic staining. Western blot analysis revealed a
ChAT-immunoreactive band of the expected size of approximately 66–70
kDa (Fig. 4D).
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). Western blotting identified a VAChT band of approximately
60 kDa, the expected size of this protein (Fig. 3E). Moreover, using
RT-PCR approaches we obtained and sequenced two partial cDNA clones
(306 bp) that proved to be identical to the published human VAChT
sequence (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regulation of steroidogenesis by human GC appears not to be a direct downstream event following activation of M1 receptors, because we did not measure changes in steroid levels (progesterone or estradiol production) when muscarinic agents were added to the cultures for 24 h at concentrations similar to those causing the observed acute rises in intracellular Ca2+ (Mayerhofer, A., S. Fritz, and C. Brucker, unpublished). However, unexpectedly, we found that these treatments affected GC growth behavior. In cultures treated with the ACh analog Carb or Oxo for 24 h, we noted that the overall protein content increased significantly. This is most likely due to an enhanced proliferation, as indicated by higher proliferation-associated PCNA levels (32) and stronger BrDU incorporation, as found in pilot studies in these cultures. This effect was further quantitated using a cell proliferation assay. Cell proliferation was linked to M1 receptor activation. Thus, the M1 receptor specificity of the effects of Oxo and Carb was indicated by the blocking effect of Pir, which abolished the observed increases. Interestingly, the antagonist Pir alone was also effective and reduced proliferation. Although the effect was not statistically significant, this gives evidence for endogenous ACh produced by cultured GC, which then may effect their growth behavior (see below). Whether apoptosis of these cells is at the same time negatively affected cannot be ruled out. However, our preliminary experiments do not indicate a major contribution of cell death in these experiments. Thus, these results indicate better survival and proliferation of GC under the treatment conditions.
Our results obtained in cultures of human GC therefore point to a growth factor-like role of the neurotransmitter ACh in human GC. Links of M1, M3, and M5 receptor to either increases (33) or decreases in mitogenesis (34) have been reported depending on the receptor subtype and the cell types examined (35). A trophic effect of ACh has previously also been proposed in the cerebellum, where ACh in the presence of nerve growth factor promoted the survival of Purkinje cells (36) or in PC12M1 cells stably expressing the M1 receptor (21). In the latter study, the muscarinic agonist Oxo at 200 µmol/L also produced morphological changes and caused the induction of marker genes for neuronal differentiation. We are currently examining whether M1 receptor activation in human GC could be linked to comparable events in this nonneuronal cell type and could also affect gene expression.
In a physiological context, linking M1 receptor activation to cell growth in GC of the ovary is of potential interest, because these cells can be regarded to represent cells derived from a preovulatory follicle undergoing luteinization and thus share characteristics with the cells of the young corpus luteum. In this endocrine compartment, numerous morphogenic events occur, including cell hypertrophy and proliferation (37). Based on our present results, we suggest that the consequences of activation of M1 receptors may contribute to these morphogenic processes in the human. Under experimental conditions in the rat (hemicastration) (38), the remaining ovary enlarges, a process in which ACh and muscarinic receptors have been implicated. Furthermore, aspects of the onset of puberty in the rat appear to be under cholinergic control (39).
It is thought that ACh is derived from the cholinergic nerve fibers found in the ovary (7, 8). However, these cholinergic ovarian nerve fibers are sparse in most species. Moreover, ACh-esterase activity of nonneuronal origin (in GC, thecal cells, corpus luteum, stroma, and smooth muscle cells) has been observed (40, 41), indicating that the half-life of ACh within the ovary may be rather short, and consequently questioning, if ACh, once released from nerve fibers, could reach potential target cells, i.e. GC within a follicle or luteal cells. In the human placenta, an organ completely devoid of innervation, there is evidence for nonneuronal expression of the ACh-synthesizing enzyme ChAT and for the presence of ACh (13, 42, 43). Gonadal ACh synthesis in the male is evidenced by ChAT expression in the human testis (12). Our present results now provide evidence for ChAT expression by endocrine cells in the human female gonad, because human GC express the necessary prerequisites of ACh synthesis ChAT and its accumulation in secretory vesicles (VAChT). Thus, expression of ChAT was detected using a monoclonal antibody in immunocytochemical experiments, in which most GC stained positive for ChAT. ChAT has been reported to exist in a multitude of as yet not completely elucidated variants due to transcriptions from distinct promoters and alternative splicing events, resulting in, for example, M, N1, N2, and R types of ChAT (14). A major ChAT isoform purified from human brain or placenta has a molecular mass of 66,000–70,000 (14, 44), a size corresponding to the ChAT form detected in the present study in Western blots of human GC. In neurons, a cholinergic cellular phenotype is determined not only by ChAT but also by the presence of an ACh-specific vesicular transporter, VAChT, which drives accumulation of ACh in secretory vesicles (24, 45). ChAT and VAChT mRNA are reported to be transcribed from the same promoter region (24) of the ChAT gene locus, indicating a unique genetic mechanism for coordinate regulation of these two cholinergic proteins. In the present study, we provide evidence that VAChT is also present in human GC using Western blotting and immunocytochemistry. These results were further substantiated by the analysis of a RT-PCR-derived cDNA, which corresponds to the human VAChT sequence (24, 45).
Our results suggest that human GC, like cholinergic neurons or nonneuronal placental syncytiotrophoblast cells or amnion epithelial cells (15), have the potential of producing and storing ACh. This also indicates that the cholinergic innervation may not represent the sole ACh source in the ovary, but that local production of ACh can also occur. Like many neurons or placental cells, human GC also possess M1 receptors, and we have shown that activation of these receptor leads to the proliferation of GC in vitro. This effect became most striking after adding exogenous M1 receptor agonist. However, it may occur also as a consequence of endogenous stimulation. Our in vitro data indicate that ACh may be produced by cultured GC and that it may affect proliferation in vitro; GC kept in culture without any treatment proliferate and incorporate BrDU (Mayerhofer, A., and S. Fritz, unpublished) and Pir, the M1 receptor antagonist, decreases the proliferation of GC. This effect, however, was not statistically significant in our experiments. Therefore, it is likely that the ACh levels produced may be relatively low in our culture system and/or that a rapid degradation may occur.
Taken together, our results suggest that a novel autocrine/paracrine regulatory loop governing cell growth may exist in human GC. Additional studies will be required to examine whether such a possibility is also realized in the in vivo counterparts of human GC cells in the ovary, i.e. in the granulosa cells of preovulatory follicles and in cells of young corpora lutea. Studies addressing this point and the regulations of M1 receptor, ChAT, and VAChT are currently being conducted in our laboratory.
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Acknowledgments |
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Footnotes |
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Received May 8, 1998.
Revised December 14, 1998.
Accepted January 21, 1999.
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References |
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-adrenergic receptor. J. Clin. Endo. Metab. 76:367–373.
(TGF) and its receptor in the hypothalamus of female rhesus
macaques. Neuroendocrinology. 60:346–359.[Medline]
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